Eukaryotic cells contain many proteases involved in cell growth, division, differentiation, migration, and intra- or extracellular signaling. One of the complicated proteolytic signaling processes is used to decide programmed cell death or apoptosis. Improper apoptosis causes many diseases including Alzheimer disease, Huntington disease, ischemia, autoimmune disorder and immortality of cancer cells [Nijhawan, D. et al. (2000) Annu. Rev. Neurosci. 23, 73-87; Rideout, H. J. & Stefanis, L. (2001) Histol. Histopathol. 16, 895-908]. For example, as cytosolic caspases play a central role in mediating the initiation and propagation of apoptosis, chemical compounds that can either inhibit or accelerate caspase activity are a major concern. Also, an understanding of the physiological proteolytic processes inside living organisms is of crucial importance for assessing the roles of proteases in normal states and diseases. Hence, the development of a rapid screening system to detect a protease activity and a noninvasive method to image apoptosis is essential for the discovery of novel compounds that are potential therapeutic chemicals [Neefjes, J. & Dantuma, N. P. (2004) Nat. Rev. Drug Discov. 3, 58-69]. These developments would provide new insights into the mechanism of apoptosis.
Several methods for monitoring protease activities have been developed; the use of peptide substrates containing a fluorescence resonance energy transfer (FRET) pair, such as fluorescein and tetramethylrhodamine, is a simple strategy for the detection of proteolytic activities [Reits, E. et al, (2003) Immunity, 18, 97-108]. D-Luciferin connected with a peptide substrate for a caspase enables highly sensitive detection of a caspase activity in vitro. However, these chemical probes do not diffuse across membranes. These methods therefore require complex assay procedures like preparation of cell lysates and elimination of sediments upon analysis of intracellular protease.
Genetically encoded fluorescent indicators that include green fluorescent protein (GFP) derivatives overcome this drawback. A potential advantage of the indicator is no need of introducing any peptide substrate into living cells [Nagai, T. & Miyawaki, A. (2004) Biochem. Biophys. Res. Commun. 319, 72-7; Takemoto, K. et al. (2003) J. Cell. Biol. 160, 235-43; Xu, X. et al. (1998) Nucleic Acids Res. 26, 2034-5; Mahajan, N. P. et al. (1999) Chem. Biol. 6, 401-9]. These indicators became valuable tools for studying temporal caspase activities in single living cells. However, the results obtained are not quantitative but rather qualitative, since the changes in the fluorescence signals are very small and the number of cells that can be analyzed is limited. In addition, it is difficult to apply the indicators to living animals because the light for excitation of the fluorophores is mostly absorbed by their tissues [Weissleder, R. & Ntziachristos, V. (2003) Nat. Med. 9, 123-8].
A recombinant bioluminescent indicator for monitoring caspase activities has been developed by using Photinus pyralis luciferase (firefly luciferase, Fluc) [Laxman B. et al. (2002) Proc. Natl. Acad. Sci. USA. 99, 16551-5]. The indicator is composed of Fluc sandwiched between estrogen-receptor regulatory domains with an Asp-Glu-Val-Asp (DEVD) sequence that can be digested by caspase-3. The usefulness of the bioluminescent indicator has been validated. That is, the activity of caspase-3 was monitored noninvasively over time in living animals. However, the molecular size of the indicator is quite large and the intensity of the luminescence signals is relatively low because of insufficient digestion of the DEVD sequence during apoptosis.
The inventors made inventions of probes binding to various target molecules in living cells as well as indicators for detecting the activities of the target molecules, utilizing the reconstruction of reporter molecules through a protein splicing with intein, and filed patent applications thereof (for example, JP-A-2004-108943; JP-A-2004-104222; JP-A-20002-088766; WO 2005/085439).